Long-range intercellular communication throughout the liver is in large part mediated by gap junction channels, through which ions and metabolites pass directly from one cell to the next. The pores of hepatocyte gap junction channels are formed primarily of the gap junction proteins connexin32 and connexin26. Recent evidence indicates that other proteins are associated with connexins at gap junctions, and we have termed this macromolecular complex the Nexus. We hypothesize that the Nexus components may regulate both the properties of the gap junction channels and also may function in intracellular signal transduction. In order to test this hypothesis, we will determine the identities of the other proteins that bind to connexins 26 and 32 in liver lysate and primary cultured hepatocytes and cell lines, measure the strengths of interaction using surface plasmon resonance and determine whether interactions are affected by post-translational connexin modifications, and perform physiological experiments with Cx32 and Cx26 mutants and binding partners to determine the functional consequences of such interactions. A major goal of this application is to obtain structural information using spectrometric methods regarding domains of Cx32 and Cx26 that interact with each other and with other proteins. These studies use a multidisciplinary approach directed at exploring a new concept in the field and as such are expected to lead to novel understanding of roles that gap junctions play in the liver and elsewhere.